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CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1β release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease. • Patients with CASP1 variants experience systemic inflammation • This occurs despite reduced caspase-1 enzymatic activity and impaired IL-1β release • CASP1 variants affect pyroptosome formation and subcellular caspase-1 localization • Variants alter pro-inflammatory cell death mechanisms and macrophage deformability • This offers previously unknown molecular mechanisms in systemic autoinflammation [ABSTRACT FROM AUTHOR]

Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein–protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders. [ABSTRACT FROM AUTHOR]